SFB796 - Sub project A3


Structural biology of plant potyvirus and cytomegalovirus effector proteins


Project summary

Protein crystallography provides for a unique and powerful tool to investigate whether common mechanisms exist for the infection of plant and mammalian cells by pathogens. The interaction between potyvirus capsid proteins and plant chaperones of the Hsp40 family has been identified as crucial in project C2 for the mechanism of potyvirus replication in plants. In phase I of CRC-796, we concentrated on the elucidation of the molecular determinants of this protein-protein interaction and succeeded in determining the first crystal structure of a plant Hsp40 protein, namely the 2.5 Å crystal structure of the substrate-binding domain of NtCPIP1. Guided by this structure and computational modelling (project A2), a substrate-binding specificity pattern was deduced for NtCPIP1, and four putative NtCPIP1 interaction sites were located within the sequence of the potyviral capsid protein of potato virus Y. Upon synthesis of the corresponding peptides, one peptide was identified that binds NtCPIP1 with high affinity (in collaboration with project A5) and that might therefore represent a major determinant for capsid-binding specificity. At present, this interaction is being corroborated in the context of the full-length proteins using extensive mutagenesis studies. Next to structures of potyvirus-derived peptide-NtCPIP1 complexes, the elucidation of the crystal structure of the complex of the full-length proteins remains still a central goal of the project since potyviruses represent a major plant virus family with no insight available today into the structure of the capsid protein. We anticipate that some of the single-site mutants that we are currently investigating will help us to resolve crystallization issues so far encountered with the native complex.

In a second part, we will study the infection mechanism of cytomegalovirus as an example for a mammalian pathogen. We so far determined the structure of the rhesus macaque cytomegalovirus major immediate early protein IE1 at 2.3 Å resolution (in collaboration with project B3). The crystal structure of IE1 displays no structural homology to any known protein structure (as judged from a Dali server inquiry). The structure is presently being refined and evaluated with various bioinformatics tools to identify putative interaction sites and to design function-deficient protein mutants (with project A2). As a next step, we will work towards the elucidation of complexes of IE1 with known host cell interaction partners such as PML.

Large scale protein production and crystallization lead to a number of crystallization hits in previous project Z. For the second phase of CRC-796 we propose that crystal structure determinations will be continued as part of present project A3. In previous project Z we obtained among others promising crystals of the human cytomegalovirus nuclear egress protein pUL50 in complex with its protein cofactor pUL53 (in collaboration with project C3) and now aim at elucidating the crystal structure of this complex, which plays a key regulatory role in the viral nuclear egress process.


Crystals of NtCPIP1, a Hsp40 protein from Nicotiana tabacum


Project relevant publications

  • Scherer M, Otto V, Stump JD, Klingl S, Müller R, Reuter N, Muller YA, Sticht H, Stamminger T.   (2015).   Characterization of recombinant human cytomegaloviruses encoding IE1 mutants L174P and 1-382 reveals that viral targeting of PML bodies perturbs both intrinsic and innate immune responses.   J. Virol. 90:1190-1205.

  • Walzer SA, Egerer-Sieber C, Sticht H, Sevvana M, Hohl K, Milbradt J, Muller YA, Marschall M.   (2015).   Crystal structure of the human Cytomegalovirus pUL50-pUL53 core nuclear egress complex provides insight into a unique assembly scaffold for virus-host protein interactions.   The Journal of Biological Chemistry 290: 27452-27458.

  • Klingl S, Scherer M, Stamminger T, Muller YA.   (2015).   Controlled crystal dehydration triggers a space-group switch and shapes the tertiary structure of cytomegalovirus immediate-early 1 (IE1) protein.   Acta Crystallographica Section D, Biological Crystallography 71: 1493-1504.

  • Scherer M, Klingl S, Sevvana M, Otto V, Schilling EM, Stump JD, Muller R, Reuter N, Sticht H, Muller YA, et al.   (2014).   Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.   PLoS Pathogens 10, e1004512.

  • Spindler N, Diestel U, Stump JD, Wiegers A-K, Winkler TH, Sticht H, Mach M, Muller YA.   (2014).   Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody.   PLoS Pathogens 10: e1004377.

  • Muller YA.   (2013).   Unexpected features in the Protein Data Bank entries 3qd1 and 4i8e: the structural description of the binding of the serine-rich repeat adhesin GspB to host cell carbohydrate receptor is not a solved issue.   Acta Cryst. Section F 69: 1071-1076.

  • Griessl MH, Schmid B, Kassler K, Braunsmann C, Ritter R, Barlag B, Stierhof Y-D, Sturm K U, Danzer C, Wagner C, Schäffer TE, Sticht H, Hensel M, Muller YA.   (2013).   Structural insight into the giant Ca2+-binding adhesin SiiE: implications for the adhesion of Salmonella enterica to polarized epithelial cells.   Structure 21: 741-752.

  • Milbradt J, Auerochs S, Sevvana M, Muller YA, Sticht H, Marschall M.   (2012).   Specific residues of a conserved domain in the N-terminus of the human cytomegalovirus pUL50 protein determine its intracellular interaction with pUL53.   J. Biol. Chem. 287: 24004-24016.

  • Sevvana M, Goetz C, Goeke D, Wimmer C, Berens C, Hillen W, Muller YA.   (2012).   An exclusive alpha/beta code directs allostery in TetR-peptide complexes.   J. Mol. Biol. 416: 46-56.

  • Griessl MH, Jungkunz I, Sonnewald U, Muller YA.   (2012).   Purification, crystallization and preliminary X-ray diffraction analysis of the Hsp40 protein CPIP1 from Nicotiana tabacum.   Acta cryst. section F 68: 236-239.

  • Sturm KU, Griessl MH, Wagner C, Deiwick J, Hensel M, Muller YA.   (2011).   Crystallization and preliminary crystallographic analysis of an Ig-domain-encompassing fragment of the giant adhesion protein SiiE from from Salmonella enterica.   Acta cryst. section F 67: 1371-1374.

  • Christoffersen C, Obinata H, Kumaraswamy S, Galvani S, Ahnström J, Sevvana M, Egerer-Sieber C, Muller YA, Hla T, Nielsen LB, Dahlbäck B.   (2011).   Endothelium-Protective Sphingosine-1-Phosphate provided by HDL-associated Apolipoprotein.   M. Proc. Natl. Acad. Sci. USA 108: 9613-9618.

  • Resch M, Schiltz E, Titgemeyer F, Muller YA.   (2010).   Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA.   Nucleic Acids Research 38: 2485-2497.

  • Stiebritz MT, Wengrzik S, Klein DL, Richter JP, Srebrzynski A, Weiler S, Muller YA.   (2010).   Computational design of a chain-specific tetracycline repressor heterodimer.   J. Mol. Biol. 403: 371-385.